Setting up a 2D-LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6-oxo-PGF(1alpha) and TXB2 as markers for hemostasis assessment.
6-oxo-PGF(1alpha) and TXB(2) are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1alpha) and TXB(2) metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1alpha), and and a LOQ of 25 pg/ml for TXB(2).
Casetta
Bruno
B
Applied Biosystems, Applera Italia, Monza, Italy. bruno.casetta@eur.appliedbiosystems.com
Vecchione
Gennaro
G
Tomaiuolo
Michela
M
Margaglione
Maurizio
M
Grandone
Elvira
E
D015121:6-Ketoprostaglandin F1 alpha / Q000097:blood*; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D004867:Equipment Design; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*; D013931:Thromboxanes / Q000097:blood*
D013931:Thromboxanes; D015121:6-Ketoprostaglandin F1 alpha